In fluorometry, excitation light on a given wavelength is directed to a sample. Illumination generates fluorescence in any fluorophore present in the sample, thus generating emission light having a longer wavelength.
If the excitation light is polarised, it will act in the fluorophore molecules settled correctly relative to the excitation light polarisation, and the emission light is polarised as well. The emission polarisation angle is specific for the fluorophore and dependent on the wavelength.
An emission may be depolarised for different reasons. Depolarisation is caused by the molecule state shifting between excitation and emission. The typical time difference is about 10 nanoseconds. Depolarisation can be used in different ways, e.g. for monitoring chemical reactions. A main cause of depolarisation is circular oscillation of a molecule. The extent of circular motion depends, among other things, on the shape and size of the molecule and on the viscosity of the medium. Depolarisation will thus be affected by the average circular motion of the molecules of the substance during the time difference between excitation and emission. If a fluorescent molecule is associated with another molecule, this will result in retarded circular motion of the fluorophore and increased polarisation of the emission.
In biotechnological applications in particular, a fluorescent fluorophore has frequently been associated with a molecule adhering specifically to identifiable molecules. An increased molecule size causes retarded circular motion of the molecule, the fluorophore thus better retaining the original polarisation level. A measurement of the polarisation thus allows direct and rapid measurement of such a specific reaction. A spectral polarisation measurement may also provide important information about the sample, involving measurement of the polarisation of the sample at different excitation or emission wavelengths.
Polarisation fluorescence is particularly suitable in the analysis of large sample quantities owing to the rapid method and the reliable measuring process.
Polarisation is measured by means of a fluorometer comprising a polarisation filter both in the excitation and the emission channel.
Two measurements are required for calculating polarisation:    1. An excitation polarisation filter and an emission polarisation filter in alignment    2. An excitation polarisation filter and an emission polarisation filter at a mutual angle of 90 degrees
The polarisation P is derived from the formula:P=(I∥−IT)/(I∥+IT)  formula (1)in which    I∥: emission intensity with parallel filters    IT: emission intensity with crossed filters
The polarisation quantity is also described with the term anisotropy r:r=(I∥−IT)/(I∥+2IT)  formula (2)
P and r can hence be calculated from each other:P(r)=3r/(2+r)  formula (1a)
Polarisation fluorometers normally use so-called L geometry, in which emission light is measured at a 90-degree angle to the excitation light. This reduces efficiently the access of excitation light to the emission detector. In the usual configuration, the excitation channel uses a stationary polarisation filter and the emission filter uses a replaceable polarisation filter, but this configuration can also be inversed.
Assumedly, the excitation channel comprises a stationary polarisation filter whose polarisation plane is X. The emission of the sample is measured with this polarisation, yielding IXX. A second measurement is performed with the emission polarisation turned by 90 degrees (plane Y), yielding Ixy. In the ideal case, the polarisation can be calculated directly from the emission measurement results IXX and IXY:P=(Ixx−IXY)/(IXX+IXY)  formula (3)
However, the transmission level of the signal from the emission channel and the transmittance of the emission polarisation filter can vary in intensity on different polarisation planes. This is why a calibration factor is necessary for compensating the difference between the measurement sensitivities of different polarisations. The calibration factor is called the G factor. The polarisation thus compensated is derived from the formula:P=(Ixx−GIXY)/(IXX+GIXY)  formula (4)
With the sensitivities of the emission measurement on different polarisation planes being SY and SX, the G factor is their mutual relationship:G=SX/SY  formula (5)
The G factor can be determined if a replaceable polarisation filter is also available on the excitation side. G is calculated by measuring the polarisation of the sample with the two excitation polarisations. Nevertheless, this manner of measuring involves numerous problems:    1. A complex design; a dual polarisation filter, requiring the filter to be positioned at an angle of exactly 90 degrees.    2. The measurement consists of four measurements and is hence slow. The G factor can be measured in advance on a suitably representative sample on a high signal level. This is awkward if the measurement is conducted on a sample whose polarisation is unknown. Replacement of polarisation filters is also a relatively slow operation considering the actual measurement period.    3. Noise; the noise of P consists of all of the four polarisation measurement components. The measurement requires a sample with adequate fluorescence for the noise factors to be minimised.
Because of the problems mentioned above, the G factor is in fact measured in most concrete cases by using a reference sample whose polarisation P is known. G can then be derived from the term of P (formula (4)). Fluoresceine, for example, is such a reference substance. This method also involves problems:    1. Maintaining the solution for any incidental measurements; fluoresceine, for instance, is apt only for applications using fluoresceine as a fluorophore.    2. Spectral polarisation measurements as a function of the excitation wavelength and the emission wavelength (P(λ)) are difficult to carry out. It is necessary to know exactly both the P(λexcitation) and the P(λemission) spectrum of the reference substance.
WO 91/07652 proposes optics and method for measuring source corrected fluorescence polarisation. In this arrangement the fluorometer relies upon a reference photodetector for monitoring the intensity of light source and for correcting the measured fluorescent intensities. In the excitation channel there is an adjustable polariser, and in the emission channel a fixed one. Only two measurements are needed for the correction: one at each position of the excitation polariser. Correction measurements may be performed with a blank cuvette or with no cuvette.